One of the most consistent findings to have emerged from psychiatric disorder genome wide association studies (GWAS) is with CACNA1C, a gene that codes for the alpha1 subunit of a voltage-dependent L-type calcium channel. Consistent with the NIMH Research Domain Criteria (RDOC) initiative, the biological implications of CACNA1C function are relevant to a diagnosis of bipolar disorder, depression, and schizophrenia. However, in spite of strong genetic data implicating sequence variations in CACNA1C as a risk factor, it is not known how genetic variants located within the gene modify risk. All GWAS-identified SNPs in CACNA1C are located within a single large intron 3 and do not lead directly to changes in the sequence of the coded protein. The central hypothesis guiding the present research effort, supported by our preliminary data, is that specific genetic variation in CACNA1C intron 3 modifies regulatory functions that can be bioinformatically predicted and experimentally validated. Our hypothesis is based on our bioinformatic analyses of the regions surrounding associated human SNPs and preliminary data from our in vitro functional validation of a subset of the in silico predictions. This proposal proposes a multiple methodology strategy, consistent with studies already underway in the laboratory. We will, in Specific Aim #1, use a bioinformatics approach to define sequences in human CACNA1C that are likely to harbor regulatory elements. We predict that the human CACNA1C gene harbors putative regulatory elements containing alleles in linkage disequilibrium (LD) with GWAS identified SNPs. Specific Aim #2 proposes to test human candidate regions in reporter vector systems to assess regulatory activity. We will clone putative regulatory elements into reporter vectors to assess their function as modifiers of transcription in vitro, fine map the location of these regulatory elements, and evaluate these elements for putative TF binding using co-transfection and TF-specific gel-shift assays. We predict that psychiatric condition-associated SNPs (or genetic variations inherited with them) will result in allele-specific changes in CACNA1C gene expression and/or altered function through cis-acting regulatory elements and that we will identify proteins that interact with such regions. As these in vitro and cell-based assays incompletely predict endogenous activity, we will map the presence of human regulatory domains in the mouse (Specific Aim #3). We will locate the corresponding regions in the mouse Cacna1c gene, validate their activity in vitro, and assess in vivo TF binding during different developmental stages. Our intent is to plan future in vivo studies with inbred or transgenic mice harboring similar genetic variations. Overall, our studies will improve basic knowledge of CACNA1C regulation, and significantly progress understanding of the mechanism by which CACNA1C gene intronic variation may modify risk for developing psychiatric disorders.